bk-1987-0325.ch005

Chapter 5

Chemical and Biological Aspects of Brassinolide Werner J . Meudt

Downloaded by PENNSYLVANIA STATE UNIV on May 22, 2013 | http://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch005

Plant Hormone Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Brassinolide (BR), (2α,3α,22α,23α-tetrahydroxy-24αmethyl-B-homo-7-oxa-5α-cholestan-6-one), a biologically active steroidal lactone first isolated from rape Brassica napus L.), pollen, stimulates growth of green plant tissues. Although the mechanism responsible for observed BR effects remains to be determined, the action of BR on growth is oligodynamic, and rapid. BR affects specific target tissues that are sensitive to the plant hormone indole-3-acetic acid (IAA)-induced growth (apparently without affecting IAA uptake and/or transport) and tissues that are gravi-perceptive. Structural analogues of BR were synthesized and the stereospecificity for its biological activity determined. Brassinosteroids thus provide plant scientists with a plant sterol for which physiological significance is demonstrated. The i s o l a t i o n of a growth stimulting chemical agent from pollen i s not new. Hans F i t t i n g , a renowned plant physiologist (1877-1970), isolated the f i r s t phytohormone, from orchid pollen (I) and recognized i t s importance i n the development of plants. F i t t i n g adopted the term "hormone" ( f i r s t proposed by E.H. S t a r l i n g i n 1905 (2)) from the medical f i e l d and introduced i t to the f i e l d of developmental physiology of plants. F i t t i n g proclaimed that " A l l e derartigen S t o f f e , d i e im eigenen Stoffwechsel des organismus Erzeugt, ohne Nahrungsstoffe zu s e i n . . . welche die Entwicklungsvorgange beeinflussen, also entwicklungsphys i o l o g i s c h von Bedeutung sind, "Hormone" zu nennen." F i t t i n g ' s attempt to i d e n t i f y h i s "Pollen Hormone" chemically was unsuccessful. Today, however, we know that pollen tissues are the source of the known plant hormones (auxin, g i b b e r e l l i n s , c y t o k i n i n ) , as well as " b r a s s i n o l i d e . " The year of F i t t i n g ' s death, 1970, was also the year that M i t c h e l l et a l (3) reported on the presence of a b i o l o g i c a l l y active agent i n rape (Brassica napus L) pollen that caused growth aberrations when applied to second internodes of 7-day o l d bean This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


54

ECOLOGY AND METABOLISM OF PLANT LIPIDS

seedlings. The growth e f f e c t observed was s u f f i c i e n t l y d i f f e r e n t from growth e f f e c t s induced by g i b b e r e l l i n s or auxins for them to suspect the presence of a new class of plant growth stimulator, which subsequently was named " b r a s s i n o l i d e " (BR). Brassinolide and s t r u c t u r a l l y related brassinosteroids have since been i s o l a t e d from various plant sources (See below). T y p i c a l l y , picomole quantities of b r a s s i n o l i d e applied to young bean seedlings w i l l cause c e l l elongation, c e l l d i v i s i o n , and s p l i t t i n g of the treated internode. The symptoms are confined to the treated area which suggests l i t t l e or no movement of the material up or down the plant axis (4). Although f i r s t i n c o r r e c t l y i d e n t i f i e d as a f a t t y acid glucoside (5) the growth response observed was primarily a t t r i b u t e d to the presence of the s t e r o i d a l lactone, brassinolide (6). Subsequent studies i n our laboratory showed that b i o l o g i c a l l y active brassinosteroids c h a r a c t e r i s t i c a l l y increases the s e n s i t i v i t y of internodal tissues of l i g h t grown bean seedlings to auxin treatments and to geotropic stimulations. Acknowledging that other auxin induced responses, such as pH changes of the incubation media (_7) and ethylene production can be increased by brassinosteroids (8,9), the e f f e c t of BR on growth i s not explicable on the basis of ethylene production or proton excretion because these e f f e c t s are also brought about by steroids other than b r a s s i n o l i d e (10) and may also occur i n mature and aged tissues that otherwise do not grow i n response to auxin, BR or geotropic stimulations. The b r a s s i n o l i d e effect i s r a p i d , oligodynamic and i s dictated by stringent s t r u c t u r a l requirements of the molecule (11) as well as by target tissue specificity. The review deals with the chemistry and physiology of brassinosteroids from which i t may be deduced that BR accelerates c e r t a i n c e l l u l a r processess that regulate the s e n s i t i v i t y of tissues to auxin and geotropic stimulations and thus may f u l f i l l a regulatory function that permits plant tissue to react to environmental (geotropism) as well as chemical (auxin) perturbations. Chemistry of Brassinolide The discovery of b r a s s i n o l i d e by M i t c h e l l et a l . about 15 years ago (3), and i t s subsequent chemical i d e n t i f i c a t i o n that showed i t to be a s t e r o i d a l B-ring lactone (6), introduced chemists to a novel s t e r o i d a l structure. C h a r a c t e r i s t i c a l l y , plant s t e r o l s contain 23 carbons comprising an unsaturated 1,2-cyclopenten-anthrene system. Joined to t h i s nucleus are angular methyl groups at carbons 10 and 13 and a nine-carbon side chain at carbon 17. The structure of b r a s s i n o l i d i s d i f f e r e n t from other phytosteroids, i n that i t s molecule i s a s t e r o i d a l lactone with the oxygen function i n the enlarged B-ring, and the s t r u c t u r a l skelton i s completely saturated. The enlarged B-ring lactone, composed of carbons 6,7,8,9, and 10, i s unprecedented i n a natural s t e r o l . In addition to the s t r u c t u r a l c h a r a c t e r i s t i c s indicated above, b i o l o g i c a l l y active brassinosteroids must also contain four hydroxyl groups, each positioned at carbons 2 and 3 of the A-ring and at carbons 22 and 23 i n the side chain. The 2,3-hydroxyl groups project to the rear (alpha) of the basic structure. The junction between rings A and B i s trans, y i e l d i n g the



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5 alpha series of compounds representing almost completely planar molecules. B i o l o g i c a l l y active brassinosteroid molecules also possess a methyl group at carbon 24, which i s also alpha- o r i e n t a t i o n . The chemical i d e n t i f y and b i o l o g i c a l a c t i v i t y has beeen v e r i f i e d through the use of synthetic b r a s s i n o l i d e . A number of b i o l o g i c a l l y active epimers and b i o l o g i c a l l y inactive isomers were used i n the study of structure a c t i v i t y r e l a t i o n s h i p s (11,12). The structure of b r a s s i n o l i d e , along with i t s three b i o l o g i c a l l y active 22,23-cis g l y c o l i c isomers, i s shown i n Figure 1 (structures I, I I , and I I I ) . The b i o l o g i c a l a c t i v i t y of the isomers i s about 50% that of brassinolide (12). A c t i v i t y i s l o s t whenever the o r i e n t i o n of the 2,3-cis g l y c o l i c groups are beta, as i n structure IV i n Figure 1. In summary the s t r u c t u r a l requirements are: a trans A/B r i n g system (alpha-hydrogen), a 6-ketone or a 7-oxa-6-ketone system i n r i n g B, c i s alpha-oriented hydroxyl groups at C-2 and C-3, c i s hydroxy groups at C-22 and C-23 as well as an a l k y l substituent at C-24. It i s of interest that the structure of brassinolide resembles that of the insect molting hormone, ecdysone, which also i s of plant o r i g i n . The structure of ecdysone i s shown i n Figure 1 and d i f f e r s from the structure of brassinolide i n that the o r i e n t a t i o n of the v i c i n a l hydroxyl group at C-2 and C-3 i s beta, the A/B junction i s c i s rather than trans as i n the brassinolide structure and that ecdysone lacks the lactone oxygen i n the B-ring. Ecdysones, that control a l l of the processes that are connected with the ecdyses of an insect are growth and d i f f e r e n t i a t i o n hormones in insects that are s i m i l a r i n function as estrogens and androgens of mammals. It i s highly conjectural, but thought provoking, that s i m i l a r i t y of structure should r e f l e c t s i m i l a r i t y i n function. I t might be suggested that brassinolides have s i m i l a r " s i g n a l " functions in plants as s t e r o i d a l hormones have i n animals by functioning as a primary chemical messenger during early events of embryogenesis and growth. Naturally Occurring Brassinosteroids

i n Plants.

Since the discovery of brassinolide i n rape pollen, 11 additional b i o l o g i c a l l y active isomers have been i s o l a t e d from higher plants. With the exception of pollen t i s s u e s , a l l of the brassinosteroids that were i s o l a t e d were found only i n young, immature, a c t i v e l y growing and d i f f e r e n t i a t i n g plant t i s s u e s , including immature seeds and shoots of a v a r i e t y of plants. The t r i v i a l and IUPAC equivalent names of brassinosteroids and related compounds are given i n Table 1. I t should be noted, with the exception of typhasterol and teasterone, that the sterochemistry of the 2,3- and 22,23- d i o l groupings are alpha oriented, as i n the brassinolide structure, and that they d i f f e r from the brassinolide structure only by the a l k y l substituent at C-24 and the degree of oxidation of the B-ring (Figure 2). A l l of the brassinosteroids l i s t e d are b i o l o g i c a l l y less active than b r a s s i n o l i d e . It i s suggested that the b i o l o g i c a l a c t i v i t y of some of these brassinosteroids might be due to t h e i r conversion to brassinolide by the plant tissue during the bioassay (15). This w i l l require further invest i g a t i o n .




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Chemical and Biobgical Aspects of Brassinolide

57


Table I . T r i v i a l and IUPAC Equivalent Names Brassinolide = (22R,23R)-2a,3a,22,23-Tetrahydroxy-24S-methyl-B-homo7-oxa-5a-cholestan-6-one I = (22S,23S)-2a,3a,22,23-Tetrahydroxy-24S-methyl-B-homo-7-oxa-5a -cho1e s t an-6-one I I = (22R,23R)-2a,3a,22,23-Tetrahydroxy-24R-methyl-B-homo-7-oxa-5a -cho1e s t an-6-one I I I = (22S,23S)-2a,3a,22,23-Tetrahydroxy-24R-methyl-B-homo-7-oxa-5a -cholest an-6-one IV = (22S,23S)-2a,3a,22,23-Tetrahydroxy-24S-ethyl-B-homo-7oxa-5a -cholestan-6-one Ecdysone = (22R)-2a,3a,14a,22,25-Pentahydroxy-5£-cholest-7-en-6-one 6-Deoxocastasterone = (22R,23R)-2a,3a,22,23-Tetrahydroxy-24S-methyl -5a-cholestane Deoxodolichosterone = (22R,23R)-2 ,3 ,22,23-Tetrahydroxy-5a -ergost-24(28)-ene 28-Norbrassinone = (22R,23R)-2a,3a,22,23-Tetrahydroxy-5a-cholestan-6 -one 28-Norbrassinolide = (22R,23R)-2a,3a,22,23-Tetrahydroxy-B-homo-7-oxa -5a-cholestan-6-one Dolichosterone = (22R,23R)-2a,3a,22,23-Tetrahydroxy-5a-ergost-24 (28)-en-6-one Dolicholide = (22R,23R)-2a,3a,22,23-Tetrahydroxy-B-homo-7-5a -ergost-24(28)-en-6-one Castasterone = (22R,23R)-2a,3a,22,23-Tetrahydroxy-24S-methyl-5a -cholestan-6-one Homodolichosterone = (22R,23R)-2a,3a,22,23-Tetrahydroxy-5a -stigmast-24(28)-en-6-one 28-Homodolichonlide = (22R,23R)-2a,3a,22,23-Tetrahydroxy-B-homo-7 -oxa-5a-st igmas t-24(28)-ene-6-one 28-Homobrassinone = (22R,23R)-2a,3 ,22,23-Tetrahydroxy-24S-ethyl-5a -cholestan-6-one 28 Homobrassinolide = (22R,23R)-2a,3a,22,23-Tetrahydroxy-24S-ethylB-homo-7-oxa-5a-cholestan-6-one Teasterone = (22R,23R)-38,22,23-Trihydroxy-24S-methyl-5a-cholestan -6-one Typhasterol = (22R,23R)-3a,22,23-Trihydroxy-24S-methyl-5a-cholestan -6-one a

a

a



58


Figure 2. Structure of n a t u r a l l y occurring brassinosteroids. T r i v i a l and IUPAC equivalent names are given i n Table I.



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Brassinolide. Brassinolide, the most b i o l o g i c a l l y active brassinos t e r i o d , was f i r s t i s o l a t e d from rape pollen (3) affording about 40 micrograms of brassinolide per kg of fresh pollen (52) . Brassinolide represents only about 0.01% of the t o t a l steroid content i n rape pollen. Rape pollen i s a r i c h source of s t e r o l i n general, c o n s t i t u t i n g about 0.1% of the fresh weight. The major steroids being 24-methylene cholesterol (13), avenasterol, c h o l e s t e r o l , and g - s i t o s t e r o l (14). Brassinolide has since been i s o l a t e d from a v a r i e t y of plants including chestnut g a l l (17,27) , inmature seeds of Chinese cabbage (15,23) and i n tea leaves (26). The concentrations are usually very low ranging from 0.25 iJg/kg (17) to 0.57 ]4g/kg (27) in chestnut g a l l tissues. In comparison, the content of castasterone in these tissues i s about f i v e times as high and that of 6-deoxocastasterone was 20 to 100-fold higher than b r a s s i n o l i d e . Small amounts of brassinolide were also detected i n inmature seeds of Chinese cabbage (15,23) and i n tea leaves (26). The amounts i n each instance were less than 10 ng/kg Fr. wt. 6-Deoxocastasterone. 6-Deoxocastasterone lacks both oxygen functions i n the B-ring of brassinolide. It i s present i n extracts of immature seeds of Phaseolus vulgaris (15), i n young insects g a l l s of chestnut trees (Castanea crenata Sieb Et Zucc.) (16) and i n healthy tissues including shoot, l e a f and flower bud of the chestnut. The larvae c o l l e c t e d from the g a l l contained at best a trace amount of a c t i v i t y which, most l i k e l y was derived from the host tissues and thus the brassinosteroid i s exclusively of plant o r i g i n . The amounts present in the various tissues range between 9 and 25 IAA) indicates that sections were f i r s t treated for 10 minutes with BR followed by IAA, (IAA—> 947-B) indicates the reverse. Sections treated with disks containing only solvent did not bend.


64


coincides with IAA treatments. No BR response i s observed when the sequence of a p p l i c a t i o n i s reversed. This assay i s s e n s i t i v e to 1.0 ng quantities of BR.


Auxin-Brassinolide Interaction The rate of change of h o r i z o n t a l displacement of the a p i c a l portion of bean f i r s t internode sections treated u n i l a t e r i a l l y i n the manner described above (bioassay) with brassinolide and/or IAA was monitored by using an angular transducer ( i n accordance with the technique described by Meudt and Bennett (37). The r e s u l t s , presented i n Figure 4, show that 0.1 nmol of IAA caused a transient growth, with a maximum rate reached about 20 minutes a f t e r auxin a p p l i c a t i o n . The start of the bending was preceded by a 10-minute l a g . Growth rates diminished gradually only to be interrupted by periodic o s c i l l a t i o n of the rates (curve A). Brassinolide lacks s i g n i f i c a n t b i o l o g i c a l a c t i v i t y of i t s own, p a r t i c u l a r l y during the f i r s t 40 minutes, a f t e r which bending does occur and reaches a maximum rate about 75 minutes a f t e r i n i t i a l start of the treatment (trace B). Since the growth pattern i n response to increasing amounts of BR does not change, we assume that the bending observed i s due to the i n t e r a c t i o n of BR and endogenous auxin rather than to brassinolide i t s e l f . When brassinolide i s applied i n combination with auxin, the auxin-induced growth i s greatly enhanced. Brassinolide apparently does not affect the f i r s t part of the growth k i n e t i c s induced by IAA. This suggests that the rate of uptake of IAA i s not affected and that b r a s s i n o l i d e has l i t t l e e f f e c t on the metabolic events that bring about the f i r s t spurt of growth induced by IAA. The change i n BR-induced growth k i n e t i c s occurs p r i m a r i l y during the second growth phase (C), i . e . , a f t e r 30 minutes. This suggest that brassinolide regulates some metabolic event that i s responsible for sustained auxin action on growth rather than i n i t i a l processes involving uptake and (or) transport. The data indicate the existence of a strong s y n e r g i s t i c i n t e r a c t i o n between brassinolide and IAA; however, follow-up experiments showed t h i s apparent synergistic r e l a t i o n s h i p does not hold true when the two treatments are applied i n c e r t a i n sequence. Results shown i n Figure 5 demonstrate that brassinolide stimulates auxin-induced growth when b r a s s i n o l i d e treatment precedes the auxin treatment ( s o l i d l i n e ) . This apparent mutual r e l a t i o n s h i p does not hold true when the sequence of a p p l i c a t i o n i s reversed (dotted l i n e ) . The growth rate of IAA pretreated sections i s not increased by a subsequent treatment with b r a s s i n o l i d e . These sections are, however, s e n s i t i z e d to auxin, as shown when the brassinolide treatment i s again replaced by IAA (dotted l i n e - 3rd h r ) . Figure 6 shows that by pretreating IAA-sensitive tissues with as l i t t l e as 10 pmol of IAA reduces the s e n s i t i v i t y of the tissues to subsequent auxin applications and that an a p p l i c a t i o n of 1 nmol of IAA desensitizes the tissues completely. This attenuation of IAA-induced growth a f t e r a chronic stimulation i s prevented by pretreating the tissues with as l i t t l e as 100 pmol of b r a s s i n o l i d e . One possible explanation of these r e s u l t s i s that the i n i t i a l application of IAA reduced the a b i l i t y of tissues to take up additional IAA by blocking putative transport channels.


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.25f

0

10

20

30

40

50

60

70

80

90

100

MIN

Figure 4. Rate of h o r i z o n t a l displacement of a p i c a l portion of bean internode sections treated with IAA (A) and BR (B) and IAA plus BR (C). 10 .

T I M E hr

Figure 5. Effect of a l t e r n a t i n g BR and IAA treatments on curvature of bean f i r s t internode sections. Sections were treated a l t e r n a t e l y either with 100 pmol of BR followed by 100 pmol of IAA ( s o l i d l i n e ) or f i r s t with IAA followed by BR (dotted l i n e ) . Treatments were exchanged at hourly i n t e r v a l s and measurements were taken at the end of each treatment.


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Pre-Treatment (nMol IAA)

0

u

1

0 0.01

1 i_L t_ 0.10 1.00 0 0.01

0 0.01 0.10 1.00

0.10 1.00 0 0.01

0.10 1.00

Post-Treatment (nMol IAA)

Figure 6. Prevention of IAA induced autogenous growth i n h i b i t i o n (bending of bean internode sections) (upper data) by BR. Bean internode section were pretreated for 1 hour with either IAA (dotted l i n e ) or IAA plus 200 pmol of BR ( s o l i d l i n e ) . Data i n lower graph show longitudinal growth of the internodes.


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Brassinolide somehow keeps these channels open, thus increasing the uptake or transport capacity of a brassinolide-treated tissue for IAA. Analysis of tissues for IAA as determined by reverse isotope d i l u t i o n assays reveals however, that b r a s s i n o l i d e does not a f f e c t auxin uptake or auxin movement within the tissues (41). In t h i s same study we also observed that bean internode sections treated with brassinolide contained s i g n i f i c a n t l y less IAA a f t e r 2 hours than tissues treated with IAA alone, even though BR potentiated IAA-induced growth by more than 400%.


Brassinolide E f f e c t on Proton Secretion The r a p i d i t y with which BR acts and i t s chemical nature suggests that membrane s i t e s associated with IAA action should be affected. Indeed, one can observe IAA-induced rapid changes of e l e c t r o p o t e n t i a l s across cytoplamsic membranes that precede c e l l enlargement, and brassinolide stimulates t h i s process (7). Unfortunately, changes i n e l e c t r o p o t e n t i a l s across cytoplasmic membranes may also be induced by steroids that are s t r u c t u r a l l y unrelated to BR (10) and do not stimulate auxin induced growth. B r a s s i n o l i d e - G i b b e r e l l i c Acid (GA) Interaction The physiological e f f e c t of b i o l o g i c a l l y active brassinosteroid on the growth of dwarf r i c e seedlings was studied by Takeno and Pharis (42), who found that nanogram quantities of b r a s s i n o l i d e increased seedling vigor and c h a r a c t e r i s t i c a l l y caused bending of the second r i c e l e a f lamina. They also pointed out that the p h y s i o l o g i c a l e f f e c t of BR i s an auxin-mediated response and that the e f f e c t i s quite d i f f e r e n t from that of GA3, which does not cause bending of the second l e a f lamina. GA3 causes marked elongation of the second r i c e l e a f sheath and i n h i b i t s BR-induced bending of the l e a f lamina. In our bioassay using the bean f i r s t internode assay (37) we observed that GA3 caused no bending of the internode but stimulated elongation of the stem section, whereas b r a s s i n o l i d e stimulated auxin-induced growth i n these t e s t s , GA3~induced growth was i n h i b i t e d (Figure 7). These r e s u l t s were confirmed by using the dwarf pea bioassay (Figure 8). In both systems, the GA response i s i n h i b i t e d by BR. Brassinolide and Ethylene Interaction Yopp et a l . (43) reported that brassinolide enhances hook closure of the bean seedling i n the dark and also enhances production of ethylene. They suggest that brassinolide actson hook closure through an effect on ethylene synthesis i n the t i s s u e s . Data from Arteca et a l . (8, 9) seem to support that brassinolide stimulated auxin-induced ethylene synthesis, p a r t i c u l a r l y i n the presence of calcium. A r e i n v e s t i g a t i o n of the possible r o l e of ethylene i n the brassinolide action on growth using bean internode sections reveal that auxininduced ethylene production i s stimulated by b r a s s i n o l i d e but as seen in Figure 9, the apparent stimulation of ethylene production can be induced i n young as well as i n mature tissues even though mature



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CURVATURE A F T E R 2hr

ELONGATION A F T E R 24hr

1.0 nmol

I IAA

NAA

1.0 nmol

2,4-D GA. |_ I A A

NAA

2 4-D t

GA

3

CONT

or

CONT

rfi

rfi

rfi

rfi rh

rfl

rfi

MINUS A N D PLUS 42.0 nmol BRASSINOLIDE Figure 7. E f f e c t of BR treatment of curvature ( l e f t ) and elongation ( r i g h t ) of bean f i r s t internode sections treated with disks containing e i t h e r 1 nmol of IAA or NAA (naphthaleneacetic-acid) or 2,4,-D (2,4-dichloro-phenoxy a c e t i c acid) or GA3 ( g i b b e r e l l i c acid) plus and minus 42 nmoles of BR.


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140.

DWARF 0 GA

3

PEA ^pM

BIOASSAY GA

3

5 pM

GA

3


120.

100.

80 B

E X

h

i O

60

40

20.

10 100

0

10

50

0

10

50

BRASSINOLIDE >ig

Figure 8. Effect of BR on GA induced growth of dwarf pea seedlings. Plants were treated with 5 p i of 0.1, or 5 JJM GA3 plus and minus 10, 50, or 100 yg of BR. Treatments were applied to the plumule of isolated seedlings. Length measurements were taken 6 days a f t e r treatment.


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tissues are not able to grow i n response to auxin or b r a s s i n o l i d e treatments. Furthermore, the increase of ethylene production i s a slow process, taking at least 4 hours, while the growth response i s rapid (observable within 60 minutes a f t e r auxin and or b r a s s i n o l i d e treatment). I interpret these r e s u l t s to mean that b r a s s i n o l i d e and auxin-induced growth involve metabolic events that are independent from auxin and BR-enhance ethylene biosynthesis.


Effect of Brassinolide or Geotropism Asymmetric growth of i s o l a t e d bean internode may be induced either by u n i l a t e r a l applications of auxin to an upright positioned section or through geostimulation by placing an i s o l a t e d internode section i n a horizontal p o s i t i o n . The l a t t e r happens i n the absence of exogenous supply of auxin. Both events are stimulated by b r a s s i n o l i d e . An example of the e f f e c t of BR on geostimulated bending of i s o l a t e d bean sections i s shown i n Figure 10. For t h i s example, the internode section were dipped for a few seconds i n aqueous s o l u t i o n of .01 ppm BR p r i o r to securing them i n a s c i n t i l a t i o n v i a l f i t t e d with a water saturated sponge and placing them i n a horizontal p o s i t i o n . The r e s u l t s indicate that BR shortens the time of graviperception of the sections which r e f l e c t an accelerated growth response. The rate of bending of control section reaches maximum about 2 hours a f t e r the beginning of geostimulation - while BR treated sections a t t a i n t h e i r maximum rate of growth i n less than one hour after geostimulation. Exogenous auxin i n t h i s system does not enhance either t h i s i n t r i n s i c geotropic response or the BR e f f e c t . Although auxin seems to have no stimulating e f f e c t on geotropic bending of i s o l a t e d bean internode sections, i t does influence the growth of p u l v i n i . Figure 11 shows that the elongation of p o l v i n i , which are the gravipercetive organs of grass shoots (44) are stimulated to grow i n response to auxin and BR a p p l i c a t i o n s . In t h i s experiment, 1.0 ug of BR or IAA was applied as l a n o l i n preparation around pieces of i s o l a t e d nodal p u l v i n i obtained either from normal or lazy corn. In both cases, IAA stimulated the growth of internodal p u l v i n i tissue and BR enhanced the auxin e f f e c t (data k i n d l y provided by Drs. P.B. Kaufman and P. Dayanandan). Effect of Brassinolide on Crop Production Brassinolide applied as a l a n o l i n preparation, a spray, or a seed treatment enhances crop production by stimulating o v e r a l l growth of plants when applied to young seedlings (45,46). T y p i c a l l y , the slow growing plants i n a population were affected more than r a p i d l y growing ones (45,46) and that i t i s affected by l i g h t . Low l i g h t i n t e n s i t i e s favor BR stimulated c e l l d i v i s o n (47,49). Crop plants grown under f i e l d conditions also benefit from brassinosteroid treatments. Lettuce, radishes, and potatoes a l l mature at an accelerated rate (50). Head lettuce seedlings, for instance, sprayed with 0.01 pm headed 2 weeks e a r l i e r than control seedlings sprayed with H2O (50). Potato tubers developed about 3 weeks e a r l i e r when plants were treated with O.lppm BR than control plants treated with just water. Plants grown under f e r t i l i z e r stress seemed to respond most favorably to BR treatment.


5. MEUDT


TOTAL ETHYLENE PRODUCTION

71

CURVATURE (mm)

36 7 DAY OLD SEEDLINGS

30 •

"

7

/TAAABR

24 •


• DAY OLO

DAY OLD

/ l / ^ ^

IAA lAAftBR 1

0 1

2

3 4 0 1 HOURS

2

3 4

Figure 9. Correlation between ethylene production ( l e f t ) and growth ( r i g h t ) of bean f i r s t internode sections from 7- and 9-day old seedlings. Treatments consisted of 1 nmol IAA and 1 g BR.

Figure 10. Effect of 0.01 ppm BR on geotropic induction of curvature of isolated bean internode sections. In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


72


TREATMENTS

Figure 11. Effect of BR on growth of normal- and Lazy Corn p u l v i n i . IAA and BR (1 g) was mixed with l a n o l i n and applied to the p u l v i n i (IMI=IAA-myo-inositol (Data of Kaufman and Dayanadan).


5.

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Encouraging r e s u l t s on the use of BR i n a g r i c u l t u r a l practices are also being generated by Hamada et a l . (53) who showed an increase i n crop y i e l d of r i c e , corn, cucumbers, sweet-potatoes etc. i n response to repeated (3x) applications of lOppm BR. BR also increased cold resistance i n egg plants and cucumbers; enhanced disease resistance against soft rot i n Chinese cabbage and against sheath b l i g h t i n r i c e plants; decreased plant i n j u r y and enhanced recovery of plants treated with various herbicides.


Conclusion C l a s s i c a l l y , plant s t e r o l s are considered either as s t r u c t u r a l constituents of cytoplasmic membranes or as precursors to animal sex hormones and insect molting hormones. It i s for that reason that i n the past, the subject of "Steroid Hormones i n Plants was of more interest to phytoendocrinologists dealing with insect molting hormones of plant o r i g i n (51) than to plant p h y s i o l o g i s t s . Steroids i n general and animal sex-hormones i n p a r t i c u l a r have, however, been tested on various plant systems with the idea that they act on sex expression of flowering of monoecious plants i n a manner s i m i l a r to t h e i r actions i n animal systems. Such evidence i s , however, very tenuous and c i r c u m s t a n t i a l . Brassinolide and b i o l o g i c a l l y active brassinosteroids provide plant s c i e n t i s t s , however, with phytosteroids to which a hormonal function i n plants can be assigned. The c e l l u l a r mechanism of BR action can at present only be reconciled with observed p h y s i o l o g i c a l e f f e c t s , and based on t h i s evidences, i t i s proposed that BR f u l f i l l s a regulatory function i n plants. The function being that BR regulates the s e n s i t i v i t y of plant tissues to auxin and to geotropic stimulation. I t i s suggested that BR somehow influences the "responsiveness" of tissues to auxin without a f f e c t i n g the i n t e r c e l l u l a r auxin concentrations. I t i s conceivable that the BR induced a m p l i f i c a t i o n of auxin action on target c e l l s involves interactions of BR with some membrane associated receptors with high a f f i n i t y for auxin and/or BR which brings auxin closer to the protein synthesis machinery of the c e l l . I t may thus function as a modulator of auxin (54) and g r a v i - s e n s i t i v e gene expression. Alternative suggestions proposed are that ethylene production (8,9) or proton secretion (7) be the biochemical locus of action of BR. In these cases BR enhanced growth ought to be explicable on the basis of either one of the chemical changes observed. Unfortunately, these BR induced biochemical changes lack the required s e l e c t i v i t y of target tissues and chemical structures s p e c i f i c i t y . Both phenomena may be induced by compounds other than brassinosteroids and also i n aged tissues that are otherwise incapable of auxin induced c e l l enlargement. 11

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January

10, 1986


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